Chemotaxis by the RAW264 mouse macrophage cell line was inhibited by 3-deazaardenosine but not by 3-deazaaristeromycin. Observations of the cells by time-lapse video photography indicated that cells treated with 3-deazaadenosine recognized the attractant and were motile, suggesting that treatment with 3-deazaadenosine inhibited signal processing. Determination of the change in intracellular levels of S-adenosylhomocysteine (AdoHcy) and 3-deazaadenosylhomocysteine (3-deaza-AdoHcy) showed that inhibition of chemotaxis was correlated with the increase in 3-deaza-AdoHcy, formed intracellularly by the utilization of 3-deazaadenosine as a substrate for AdoHcy hydrolase. The synthesis of phosphatidylcholine by stepwise methylation of phosphatidylethanolamine, the attractant-induced release of arachidonic acid, methylation of lysine and arginine residues in protein, and carboxymethylation were inhibited by both 3-deazaadenosine and 3-deazaaisteromycin, indicating that these reactions were not required for chemotaxis. In addition, cAMP levels were not significantly changed in cells incubated with 3-deazaadenosine or 3-deazaaristeromycin. However, the synthesis of a small number of proteins, separated by two-dimensional polyacrylamide gel electrophoresis, was susceptible to 3-deazaadenosine, but not to 3-deazaaristeromycin. Quantitation of the gels by computerized densitometry showed that the synthesis of approximately 10% of the proteins was inhibited by more than 50%. A correlation was found between inhibition of chemotaxis and inhibition of the synthesis of the same subset of proteins when other compounds were tested. These compounds also inhibited the synthesis of polyadenylated RNA, leading us to postulate that incubation of cells with 3-deazaadenosine inhibits a methylation reaction that is required for the formation of a functional mRNA coding for one or more proteins required for chemotaxis.